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• Site- and structure-specific identification of N-glycosylation of Arabidopsis at the intact N-glycopeptide level. • With spectrum-level FDR ≤ 1%, 5,687 intact N-glycopeptides coming from 3,713 N-glycosites of 3,140 N-glycoproteins were identified. • With structure-diagnostic fragment ions as experimental evidence, monosaccharide sequence structures of N-glycans on 630 intact N-glycopeptides were unambiguously distinguished from the other sequence isomers with the same monosaccharide compositions. The N-glycosylation is an important bioprocess in plant. Monosaccharide composition-level characterization at the intact N-glycopeptides has been extensively reported, yet structure-specific study to resolve multiple sequence structures of a single composition is still lacking. Here, we present a comprehensive structure-specific identification of intact N-glycopeptides of Arabidopsis with both HILIC and RAX enrichment, as well as GPSeeker and pGlyco database search. With target-decoy searches and spectrum-level FDR ≤ 1%, 5,687 N-glycopeptides from 3,713 N-glycosites of 3,140 N-glycoproteins were identified, which represents the currently most comprohensive profilling to our best knowledge. Wtih the experimental evidence support of structure-diagnostic fragment ions, 81 glycan structures from 54 glcan compostions were unambiguouly distinguished. The comprehensive experimental site- and structure-specific N-glycosylation data reported in this study will serve as a fundamental valuable reference for the coming functional studies of this widely adopted model organism of plant. [ABSTRACT FROM AUTHOR]

[Display omitted] • 2764 IDs distinguished from sequence isomers by structure-diagnostic fragment ions. • With fold change ≥1.5 and p value < 0.05, 104 DEGPs were determined. • ITGAV, ITGB5 and LAMA1 were associated with ECM-receptor interaction by KEGG analysis. • APC, DVL3 and INVS were associated with Wnt signaling pathway by KEGG analysis. • Down-regulated intact N-glycopeptide with bisecting GlcNAc from SPT20H were found. • Upregulated intact N-glycopeptide with β1,6-branching N-glycan from ITGB5 were found. Breast cancer is responsible for the highest mortality all over the world. Cancer stem cells (CSCs) along with epithelial mesenchymal transition (EMT) are identified as a driver of cancer which are responsible for cancer metastasis and drug resistance. Several signaling pathways are associated with drug resistance. Additionally, glycosyltransferases regulate different types of glycosylation which are involved in drug resistance. To the end, it is urgent to figure out the knowledge on cell-surface altered N-glycosylation and putative markers. Here, differential cell-surface intact N-glycopeptides in adriamycin (ADR)-resistant michigan breast cancer foundation-7 stem cells (MCF-7/ADR CSCs) relative to ADR-sensitive MCF-7 CSCs were analyzed with site- and structure-specific quantitative N-glycoproteomics. The intact N-glycopeptides and differentially expressed intact N-glycopeptides (DEGPs) were determined and quantified via intact N-glycopeptide search engine GPSeeker. Totally, 4777 intact N-glycopeptides were identified and N-glycan sequence structures among 2764 IDs were distinguished from their isomers by structure-diagnostic fragment ions. Among 1717 quantified intact N-glycopeptides, 104 DEGPs were determined (fold change ≥ 1.5 and p value < 0.05). Annotation of protein–protein interaction and biological processes among others of DEGPs were finally carried out; down-regulated intact N-glycopeptide with bisecting GlcNAc from p38-interacting protein and up-regulated intact N-glycopeptide with β1,6-branching N-glycan from integrin beta-5 were found. [ABSTRACT FROM AUTHOR]